Structure‐Based Design and Synthesis of a Small Molecule that Exhibits Anti‐inflammatory Activity by Inhibition of MyD88‐mediated Signaling to Bacterial Toxin Exposure

Abstract

Both Gram-positive and Gram-negative pathogens or pathogen-derived components, such as staphylococcal enterotoxins (SEs) and endotoxin (LPS) exposure, activate MyD88-mediated pro-inflammatory cellular immunity for host defense. However, dysregulated MyD88-mediated signaling triggers exaggerated immune response that often leads to toxic shock and death. Previously, we reported a small molecule compound 1 mimicking BB-loop structure of MyD88 was capable of inhibiting pro-inflammatory response to SEB exposure in mice. In this study, we designed a dimeric structure compound 4210 covalently linked with compound 1 by a non-polar cyclohexane linker which strongly inhibited the production of pro-inflammatory cytokines in human primary cells to SEB (IC50 1-50 μm) or LPS extracted from Francisella tularensis, Escherichia coli, or Burkholderia mallei (IC50 10-200 μm). Consistent with cytokine inhibition, in a ligand-induced cell-based reporter assay, compound 4210 inhibited Burkholderia mallei or LPS-induced MyD88-mediated NF-kB-dependent expression of reporter activity (IC50 10-30 μm). Furthermore, results from a newly expressed MyD88 revealed that 4210 inhibited MyD88 dimer formation which is critical for pro-inflammatory signaling. Importantly, a single administration of compound 4210 in mice showed complete protection from lethal toxin challenge. Collectively, these results demonstrated that compound 4210 inhibits toxin-induced inflated pro-inflammatory immune signaling, thus displays a potential bacterial toxin therapeutic.

Rational design of peptide derivatives for inhibition of MyD88‐mediated toll‐like receptor signaling in human peripheral blood mononuclear cells and epithelial cells exposed to Francisella tularensis

“…Compounds 4 – 56 were tested for inhibition of cytokines in primary human peripheral blood mononuclear cells (hPBMCs) treated with F. tularensis LPS extracts (10 μg/ml) using an adaptation of protocols previously described for other pathogens . Cells were preincubated with test compound for 30 min and then exposed to LPS extracts from F. tularensis (10 μg/ml) for 16 hr.…”

“…Cells were preincubated with test compound for 30 min and then exposed to LPS extracts from F. tularensis (10 μg/ml) for 16 hr. Cytokine response was measured using a MSD human proinflammatory fourplex ultrasensitive kit (TNF‐α, IFN‐γ, IL‐6, IL‐1β) with electrochemiluminescent output . The calibrated response curve demonstrated that all four cytokines gave a linear response in the range of 1 pg/ml to 10 μg/ml of LPS extracts.…”

“…Compound ST2825 was reported to be based on a heptapeptide sequence of this domain and blocked TIR domain interactions induced by IL‐1β treatment and reduced IL‐6 levels in C57BL/6 mice. Additionally, we have reported a dimerization strategy for AS1 that gave compounds (i.e., EM163 and 4210 ) that were significantly more potent than AS1 , and compound 4210 attenuated proinflammatory cytokines induced by several biothreat agents and was efficacious in a lethal shock challenge in BALB/c mice . More recently, ortho ‐vanillin and certain derivatives were identified as inhibitors of the TIR domain interactions of TLR2 and inhibited TLR2‐mediated inflammation in mice .…”

“…Direct comparisons of potency among compound 7 and the above TIR domain inhibitors are difficult due to the different experimental conditions and proinflammatory stimulants used, but compound 7 appears to have comparable potency range as most of the larger peptide derivatives and ortho ‐vanillin (i.e., NF‐κb reporter data indicated inhibition in a range of 1–200 μ m for most molecules where data are available). Dimeric AS1 analogs such as 4210 have been shown to be a strategy for increasing the potency of these small tripeptide mimetics . Thus, the potency of compound 7 relative to size of the molecule represents a possibly advantageous property.…”

“…AS1 was effective at blocking proinflammatory cytokines in response to IL‐1 receptor activation . Dimeric analogs of AS1 were also developed that more potently disrupt protein–protein interactions of MyD88 . AS1 attenuated in vitro IL‐1β signaling in murine lymphocytes and EL4 thymoma cells in a concentration range of 10–100 μ m and reduced IL‐1β‐induced fever in mice at 200 mg/kg (i.p.).…”